The present invention relates to identification of the consensus sequence phosphorylated by ATM kinase. This, in turn, permitted identification of ATM kinase target proteins, and development of a convenient assay system for ATM kinase phosphorylation using fusion polypeptides as substrates. The assay system is adaptable to screening for ATM modulators, particularly inhibitors.
Ataxia telangiectasia (AT) is a rare autosomal recessive multi-system disorder characterized by clinical manifestations that include progressive cerebellar ataxia, neuronal degeneration, hypersensitivity to ionizing irradiation (IR), premature aging, hypogonadism, growth retardation, immune deficiency, and an increased risk for cancer (Lavin and Shiloh, Annu. Rev. Immunol., 15:177, 1997). Cancer predisposition in AT is striking: 38% of patients develop malignancies, mainly lymphoreticular neoplasms and leukemias. But AT patients manifest acute radiosensitivity and must be treated with reduced radiation doses, and not with radiomimetic chemotherapy. AT has a worldwide frequency of 1:100,000 live births and an estimated carrier frequency of 1% in the American population. Notable concentrations of AT patients outside the United States are in Turkey, Italy, and Israel.
Cerebellar ataxia that gradually develops into general motor dysfunction is the first clinical hallmark and results from progressive loss of Purkinje cells in the cerebellum. Oculocutaneous telangiectasia (dilation of blood vessels) develops in the bulbar conjunctiva and facial skin, and is later accompanied by graying of the hair and atrophic changes in the skin. Somatic growth is retarded in most patients, and ovarian dysgenesis is typical for female patients. Among occasional endocrine abnormalities, insulin-resistant diabetes is predominant, and serum levels of alpha-fetoprotein and carcinoembryonic antigen are elevated. The thymus is either absent or vestigial, and other immunological defects include reduced levels of serum IgA, IgE or IgG2, peripheral lymphopenia, and reduced responses to viral antigens and allogeneic cells. These immunological defects cause many patients to suffer from recurrent sinopulmonary infections. The most common cause of death in AT, typically during the second or third decade of life, is from these sinopulmonary infections with or without malignancy.
The gene mutated in AT, ATM (Ataxia Telangiectasia-Mutated), encodes a 370-kD protein that is a member of a family of proteins related to phosphatidylinositol 3-kinase (PI-3-K) that have either lipid or protein kinase activity. A subset of this family with the greatest homology to ATM functions in DNA repair, DNA recombination, and cell cycle control (Savitsky et al., Science, 268:1749, 1995; Keith and Screiber, ibid., 270:50, 1995). Cell lines derived from AT patients exhibit hypersensitivity to ionizing radiation (IR) and defects in several IR-inducible cell cycle checkpoints, including a diminished irradiation-induced arrest in the G1 phase of the cell cycle mediated by the p53 tumor suppressor gene product (Kastan et al., Cell, 71:587, 1992; Morgan and Kastan, Adv. Cancer Res., 71:1, 1997). In response to DNA damage, cells with wild type ATM accumulate p53 protein and show a subsequent increase in p53 activity, whereas cells with defective ATM show a smaller increase in the amount of p53 protein in response to IR (Kastan et al., supra; Canman et al., Cancer Res., 54:5054, 1994; Khanna and Lavin, Oncogene, 8:3307, 1993). Therefore, ATM appears to act upstream of p53 in a signal transduction pathway initiated by IR.
p95/nibrin has recently been shown to be the gene mutated in the Nijmegen breakage syndrome (NBS), which is an autosomal recessive disease with a phenotype (radiation sensitivity, predisposition to malignancies, and chromosomal instability) virtually identical to that of AT (Shiloh, 1997; Carney et al., 1998; Varon et al., 1998; Featherstone and Jackson, 1998). The main distinction between the AT and NBS syndromes is that AT patients exhibit progressive ataxia while NBS patients have microcephaly as their neurologic abnormality. p95/nibrin is part of a double-strand break DNA repair protein complex containing Rad50 and MRE11 (Carney et al., 1998; Dong et al., 1999; Paul and Gellert, 1999). The overlapping phenotypes of AT and NBS suggest that ATM and p95/nibrin may play a role in the same cellular pathways, likely including roles in responses to DNA damage. Identifying p95/nibrin as an in vitro ATM kinase target led us to investigate potential in vivo interactions between p95/nibrin and ATM. We found that ATM protein can bind to p95/nibrin and that ATM activity is required for phosphorylation of p95 on Ser343 after IR. Thus, characterization of the ATM kinase and elucidation of in vitro targets led to identification of one valid physiologically significant target and will likely lead to further insights into ATM function and AT biology.
IR induces rapid, de novo phosphorylation of endogenous p53 at two serine residues within the first 24 amino acids of the protein, one of which was identified as Ser15 (Shieh et el., Cell, 91:325, 1997; Siliciano et al., Genes Dev., 11:3471, 1997). Phosphorylation of p53 at Ser15 in response to DNA damage correlates with both the accumulation of total p53 protein as well as with the ability of p53 to transactivate downstream target genes in wild type cells (Siliciano et al., supra). Furthermore, phosphorylation of p53 on Ser15 in response to IR is diminished in cell lines derived from AT patients, suggesting that ATM participates in this response (Siliciano et al., supra).
The PI3-K-related protein, DNA-activated protein kinase (DNA-PK), phosphorylates p53 in vitro at two different SQ motifs, Ser15 and Ser37 (Lees-Miller et al., Mol. Cell Biol., 12:5041, 1992). However, cells with diminished DNA-PK activity still normally accumulate p53 protein and undergo G1 arrest in response to IR (Rathmell et al., Cancer Res., 57:68, 1997; Guialos et al., Genes Dev., 10:2038, 1996; Nacht et al., ibid., p. 2055).
The concept of inhibiting ATM for the treatment of neoplasms, particularly cancers associated with decreased p53 function, has been suggested (Morgan et al., Mol. Cell Biol. 17:2020, 1997; Hartwell and Kastan, Science, 266:1821, 1994; Kastan, New Eng. J. Med. 333:662, 1995; see also WO 98/56391, Westphal and Leder). In particular, Westphal and Leder provide genetically manipulated knock-out mice as a model for testing ATM inhibitors. This published application suggests using an inhibitory antibody to ATM, a dominant negative fragment of ATM (see also Morgan et al., Supra), or an ATM antisense strategy to inhibit ATM. However, while these publications propose inhibiting ATM to enhance radiosensitivity of neoplastic cells, and screening for compounds that inhibit ATM activity, they provide no specific screening test, particularly one suitable for high through-put screening. There is no hint or suggestion in these publications of strategies for targeting the kinase activity of ATM, the nature of an ATM kinase substrate recognition sequence, or of sequences recognized specifically by ATM, but not other kinases. There is also no information about the function of other ATM target proteins besides p53.
Accordingly, there is a need in the art to understand ATM kinase specificity. There is a further need to identify ATM target proteins other than p53.
These and other needs in the art are addressed by the present invention.
The present invention relates to the identification of an ATM kinase substrate recognition consensus sequence motif, and to the identification of new ATM target proteins, which in turn has led to the discovery of unexpected and novel ATM-regulated cellular pathways.
Thus, in one embodiment, the invention advantageously provides a method for identifying an ATM kinase substrate recognition sequence in a protein. This method comprises contacting an ATM kinase with a fusion polypeptide and detecting whether binding has occurred between the ATM kinase and the fusion polypeptide. The fusion polypeptide contains a structural portion and a candidate ATM-kinase substrate recognition sequence portion. Moreover, given application of sequence comparison techniques, the invention provides a method for identifying a putative ATM target protein, by analyzing the sequence of the protein to determine whether it contains an ATM substrate recognition consensus sequence motif.
In a further embodiment, a method for identifying an ATM-regulated pathway is provided. This method comprises identifying a substrate of an ATM kinase, e.g., as described above; modulating ATM-mediated phosphorylation of the target protein comprising an ATM recognition sequence; and determining whether modulation of ATM-mediated phosphorylation of the target protein affects a cellular pathway, which would indicate that the pathway is an ATM-regulated pathway. As a corollary, the invention provides a method for modulating an ATM-regulated pathway, which comprises modulating ATM-mediated phosphorylation of a target protein comprising an ATM-kinase recognition sequence in a cell.
The methods of the invention can involve a kinase-dead ATM mutant polypeptide. Thus, in a further embodiment, the invention provides a nucleic acid encoding such a polypeptide, as well as the polypeptide itself. The invention provides a recombinant vector which codes for expression of a defective ATM polypeptide, e.g., a kinase dead mutant, and a cell line containing such a vector.
In another embodiment, particularly in connection with methods for identifying an ATM kinase substrate recognition sequence and for screening, the invention provides a fusion polypeptide, wherein the fusion polypeptide contains a structural portion and an ATM-kinase recognition sequence portion.
In still another embodiment, the invention provides a method for screening for a compound that modulates ATM-mediated phosphorylation. This method comprises detecting whether there is a change in the level of ATM-mediated phosphorylation of a polypeptide comprising an ATM substrate recognition sequence in the presence of a candidate compound, wherein an increase in the level of phosphorylation indicates that the compound agonizes ATM-mediated phosphorylation, and a decrease in the level of phosphorylation indicates that the compound antagonizes ATM-mediated phosphorylation.
The invention further provides for screening for a compound that induces an ATM-regulated pathway in a cell, comprising contacting the cell with a candidate compound, and detecting whether the ATM-mediated pathway is induced in the cell, wherein the cell is defective for expression of ATM. In one embodiment, the screening can be for ATM-regulated cellular pathway, with the proviso that the pathway does not involve p53 or cell cycle control, or both.
The invention also provides methods for modulating ATM kinase activity in cells in vitro and in vivo. Such modulation includes inhibition of ATM kinase. In vivo modulation provides for evaluation of ATM function, e.g., in animal models. Alternatively, in vivo modulation of ATM function has therapeutic effects. In particular, ATM inhibition can enhance radiosensitivity and chemotherapeutic sensitivity in tumors, inhibit cell proliferation and induce revascularization in restenosis, and promote insulin signaling and increased metabolism in obesity.
In yet another embodiment, the invention provides a composition comprising ATM and a polypeptide, in which the polypeptide comprises an ATM kinase substrate recognition sequence, e.g., for co-crystallization or other methods of structure-function analysis. The results of such structural studies permit rational drug design and development.
These and other aspects of the present invention are further elaborated in the Detailed Description of the Invention and Examples, infra.